Compositions and methods of using chondroitinase abci mutants

ABSTRACT

The present disclosure relates to protein and nucleic acid mutants of chondroitinase ABCI. Such nucleic acid mutants encode for chondroitinase ABCI mutant enzymes exhibiting altered chondroitin lyase activity or increased resistance to inactivation from stressors including UV light or heat. Methods of using such nucleic acid mutants encoding chondroitinase ABCI mutant enzymes is also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/790,075, filed Jul. 2, 2015, which is a divisional application ofU.S. patent application Ser. No. 13/849,420, filed Mar. 22, 2013, nowU.S. Pat. No. 9,102,930, issued Aug. 11, 2015, which is continuationapplication of U.S. patent application Ser. No. 12/781,762, filed May17, 2010, now U.S. Pat. No. 8,404,232, issued Mar. 26, 2013, which is adivisional application of U.S. patent application Ser. No. 11/870,350,filed Oct. 10, 2007, now U.S. Pat. No. 7,722,864, issued May 25, 2010,which claims the benefit of U.S. Provisional Patent Application No.60/828,800 filed Oct. 10, 2006, the contents of each being incorporatedby reference as if set forth in their entirety herein.

BACKGROUND

The spinal cord is made up of nerve fibers. Damage to the centralnervous system, including the spinal cord, results in a loss offunction. The most common types of spinal cord injuries (SCI) includecontusions (bruising of the spinal cord) and compression injuries(caused by prolonged pressure on the spinal cord). After a spinal cordinjury in the adult mammal, the inability of axons to regenerate maylead to loss of sensation, loss of motor function and/or loss ofautonomic function, as well as permanent paralysis. One reason thatneurons fail to regenerate is their inability to traverse the glial scarthat develops following a spinal cord injury. The injury-induced lesionwill develop glial scarring, which contains extracellular matrixmolecules including chondroitin sulfate proteoglycans (CSPGs). CSPGsinhibit nerve tissue growth in vitro and nerve tissue regeneration atCSPGs rich regions in vivo. CSPGs are implicated in various otherconditions including, for example, inflammation.

SUMMARY OF THE INVENTION

One embodiment of the present invention provides mutants of achondroitinase ABCI enzyme.

In preferred embodiments, such chondroitinase ABCI mutant enzymesexhibit enhanced activity. In other preferred embodiments, suchchondroitinase ABCI mutant enzymes exhibit enhanced resistance toinactivation, including inactivation from UV or heat exposure.

Preferably, a chondroitinase ABCI mutant enzyme of the invention isselected from 055D2-3 (SEQ ID NO:1), 079B6-2 (SEQ ID NO:2), 079D2-2 (SEQID NO:3), 057G1-1 (SEQ ID NO:4), 023G6-4 (SEQ ID NO:5) 005B12-3 (SEQ IDNO: 6), and 021B8-3 (ATCC Deposit Designation PTA-8657).

The nucleotide sequence of wild type chondroitinase ABCI of Proteusvulgaris is set forth as SEQ ID NO:7 and the amino acid sequence ofchondroitinase ABCI is set forth as SEQ ID NO:8.

The invention includes nucleic acids encoding the chondroitinase ABCImutant enzymes of the invention and methods of their use. In anembodiment, the invention includes a nucleic acid sequence that encodesa chondroitinase ABCI mutant enzyme selected from 055D2-3 (SEQ ID NO:1),079B6-2 (SEQ ID NO:2), 079D2-2 (SEQ ID NO:3), 057G1-1 (SEQ ID NO:4),023G6-4 (SEQ ID NO:5) 005B12-3 (SEQ ID NO: 6), and 021B8-3 (ATCC DepositDesignation PTA-8657). Preferably, a nucleic acid sequence of theinvention is selected from 055D2-3 nucleic acid (SEQ ID NO:9), 079B6-2nucleic acid (SEQ ID NO:10), 079D2-2 nucleic acid (SEQ ID NO:11),057G1-1 nucleic acid (SEQ ID NO:12), 023G6-4 nucleic acid (SEQ ID NO:13)005B12-3 nucleic acid (SEQ ID NO: 14), and 021B8-3 (ATCC DepositDesignation PTA-8657).

Other embodiments of the present invention relate to methods of treatinga patient in need of neurological functional recovery, includingsensory, motor and autonomic function, after, for example, centralnervous system (“CNS”) injury or disease. The ABCI mutant enzymes of theinvention can also be used to degrade CSPGs. Accordingly, an embodimentof the invention includes a method of degrading one or more CSPGs usinga composition comprising an ABCI mutant enzyme of the invention.Preferably a composition of the invention effective for promotingneurological functional recovery comprises a chondroitinase ABCI mutantenzyme selected from 055D2-3 (SEQ ID NO:1), 079B6-2 (SEQ ID NO:2),079D2-2 (SEQ ID NO:3), 057G1-1 (SEQ ID NO:4), 023G6-4 (SEQ ID NO:5)005B12-3 (SEQ ID NO: 6), and 021B8-3 (ATCC Deposit DesignationPTA-8657).

One embodiment of the present invention is a method for modifying accessof cells to extravascular spaces and regions, comprising administeringto a patient a composition comprising an enzyme of the invention.Another embodiment of the present invention is a method of reducingpenetration of cells associated with inflammation into tissue of apatient. Preferably, the enzyme is selected from 055D2-3 (SEQ ID NO:1),079B6-2 (SEQ ID NO:2), 079D2-2 (SEQ ID NO:3), 057G1-1 (SEQ ID NO:4),023G6-4 (SEQ ID NO:5) 005B12-3 (SEQ ID NO: 6), and 021B8-3 (ATCC DepositDesignation PTA-8657).

Another embodiment of the present invention is a method for inhibitingextravasation of cells associated with inflammation from blood vessels,comprising administering to a patient a composition comprising an enzymethat cleaves chondroitin sulfate proteoglycans. In an embodiment, anenzyme of the invention prevents cells selected from the groupconsisting of white blood cells, leukocytes, neutrophils, eosinophils,basophils, lymphocytes, B-cells, T-cells, monocytes, and macrophagesfrom leaving the blood stream. Preferably, the enzyme is selected from055D2-3 (SEQ ID NO:1), 079B6-2 (SEQ ID NO:2), 079D2-2 (SEQ ID NO:3),057G1-1 (SEQ ID NO:4), 023G6-4 (SEQ ID NO:5) 005B12-3 (SEQ ID NO: 6),and 021B8-3 (ATCC Deposit Designation PTA-8657).

Another embodiment of the invention is a method of treating inflammationin a patient comprising administering to the patient an enzyme thatcleaves chondroitin sulfate proteoglycans. Preferably, the enzyme isselected from 055D2-3 (SEQ ID NO:1), 079B6-2 (SEQ ID NO:2), 079D2-2 (SEQID NO:3), 057G1-1 (SEQ ID NO:4), 023G6-4 (SEQ ID NO:5) 005B12-3 (SEQ IDNO: 6), and 021B8-3 (ATCC Deposit Designation PTA-8657). In variousembodiments of the present invention, inflammation is associated withdisease or injury, such as chronic inflammatory disease and centralnervous system disease.

Another embodiment of the invention is a method of preventinginflammation in a patient, comprising administering to the patient acomposition comprising an enzyme that cleaves chondroitin sulfateproteoglycans. Preferably, the enzyme is selected from 055D2-3 (SEQ IDNO:1), 079B6-2 (SEQ ID NO:2), 079D2-2 (SEQ ID NO:3), 057G1-1 (SEQ IDNO:4), 023G6-4 (SEQ ID NO:5) 005B12-3 (SEQ ID NO: 6), and 021B8-3 (ATCCDeposit Designation PTA-8657).

Another embodiment of the present invention is a method of treatinginflammation in a patient, comprising extracting cells associated withinflammation from a patient, subjecting the cells to an enzyme thatcleaves chondroitin sulfate proteoglycans ex vivo to modify the cells,and administering the modified blood cells into the patient. Preferably,the enzyme is selected from 055D2-3 (SEQ ID NO:1), 079B6-2 (SEQ IDNO:2), 079D2-2 (SEQ ID NO:3), 057G1-1 (SEQ ID NO:4), 023G6-4 (SEQ IDNO:5) 005B12-3 (SEQ ID NO: 6), and 021B8-3 (ATCC Deposit DesignationPTA-8657).

In an embodiment, an enzymes of the present invention is used to treat apatient in need of regeneration of damaged neurological tissue. Inanother embodiment, an enzyme of the invention is used to facilitatediffusion and transport of therapeutic molecules capable of blockingand/or overcoming the activity of neuronal growth inhibitory moleculesinto damaged or diseased tissue. Embodiments of the present inventioninclude compositions comprising chondroitinase ABCI mutant enzymes ofthe invention and methods for their use to facilitate delivery anddiffusion of therapeutics or diagnostic agents, and agents that promoteregeneration of nerves and axons, into cells or tissues. Preferably acomposition of the invention is effective in the regeneration of damagedneurological tissue or to facilitate diffusion or transport. In anembodiment, a composition of the invention comprises a chondroitinaseABCI mutant enzyme selected from 055D2-3 (SEQ ID NO:1), 079B6-2 (SEQ IDNO:2), 079D2-2 (SEQ ID NO:3), 057G1-1 (SEQ ID NO:4), 023G6-4 (SEQ IDNO:5) 005B12-3 (SEQ ID NO: 6), and 021B8-3 (ATCC Deposit DesignationPTA-8657).

Further embodiments relate to methods of promoting neuronal outgrowthand use in treating spinal cord injuries and related disorders of theCNS by administering such a chondroitinase ABCI mutant enzyme.Preferably a composition of the invention effective for promotingneuronal outgrowth comprises a chondroitinase ABCI mutant enzymeselected from 055D2-3 (SEQ ID NO:1), 079B6-2 (SEQ ID NO:2), 079D2-2 (SEQID NO:3), 057G1-1 (SEQ ID NO:4), 023G6-4 (SEQ ID NO:5) 005B12-3 (SEQ IDNO: 6), and 021B8-3 (ATCC Deposit Designation PTA-8657).

DESCRIPTION OF THE DRAWINGS

In part, other aspects, features, benefits and advantages of theembodiments of the present invention will be apparent with regard to thefollowing description, appended claims and accompanying drawings where:

FIG. 1 shows relative mutant chondroitinase protein levels in whole celllysates, as more fully described below in Example 3.

FIG. 2 shows the results of a stability assay of mutant chondroitinaseABCI whole cell lysates at 37° C., as more fully described in Example 3.

FIG. 3 shows the results of a stability assay of semi-purified mutantchondroitinase ABCI enzymes, as more fully described in Example 4.

DETAILED DESCRIPTION

Before the present compositions and methods are described, it is to beunderstood that this invention is not limited to the particularmolecules, compositions, methodologies or protocols described, as thesemay vary. It is also to be understood that the terminology used in thedescription is for the purpose of describing the particular versions orembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims.

It must also be noted that as used herein and in the appended claims,the singular forms “a,” “an,” and “the” include plural reference unlessthe context clearly dictates otherwise. Thus, for example, reference toa “cell” is a reference to one or more cells and equivalents thereofknown to those skilled in the art, and so forth. Unless definedotherwise, all technical and scientific terms used herein have the samemeanings as commonly understood by one of ordinary skill in the art.Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of embodimentsof the present invention, the preferred methods, devices, and materialsare described. All publications mentioned herein are incorporated byreference. Nothing herein is to be construed as an admission that theinvention is not entitled to antedate such disclosure by virtue of priorinvention.

As used herein, the term “about” means plus or minus 10% of thenumerical value of the number with which it is being used. Therefore,about 50% means in the range of 45%-55%.

“Administering” when used in conjunction with a therapeutic means toadminister a therapeutic directly into or onto a target tissue or toadminister a therapeutic to a patient whereby the therapeutic positivelyimpacts the tissue to which it is targeted. Thus, as used herein, theterm “administering,” can include, but is not limited to, providing anenzyme into or onto a target tissue; providing an enzyme systemically toa patient by, e.g., intravenous injection whereby the therapeuticreaches the target tissue; providing an enzyme in the form of theencoding sequence thereof to the target tissue (e.g., by so-calledgene-therapy techniques).

The term “animal” as used herein includes, but is not limited to, humansand non-human vertebrates such as wild, domestic and farm animals.

The term “improves” is used to convey that the present invention changeseither the appearance, form, characteristics and/or the physicalattributes of the target to which it is being provided, applied oradministered. The change may be demonstrated by any of the followingalone or in combination, including degradation of the CSPGs of thelesioned area of the spinal cord or within the CNS or restoring, inwhole or in part, motor, sensory or autonomic function of the mammal.

The term “inhibiting” includes administering a compound of the presentinvention to prevent the onset of the symptoms, alleviating thesymptoms, or eliminating the disease, condition or disorder.

By “pharmaceutically acceptable,” it is meant the carrier, diluent orexcipient must be compatible with the other ingredients of theformulation and not deleterious to the recipient thereof

The term “recombinant protein” refers to a polypeptide of the presentinvention which is produced by recombinant DNA techniques, whereingenerally, DNA encoding a polypeptide is inserted into a suitableexpression vector which is in turn used to transform a host cell toproduce the protein. Moreover, the phrase “derived from,” with respectto a recombinant gene, is meant to include within the meaning of“recombinant protein” those proteins having an amino acid sequence of anative protein, or an amino acid sequence similar thereto which isgenerated by mutations including substitutions and deletions (includingtruncation) of a naturally occurring form of the protein.

As used herein, the term “therapeutic” means an agent utilized to treat,combat, ameliorate, prevent or improve an unwanted condition or diseaseof a patient. In part, embodiments of the present invention are directedto the treatment of the central nervous system, such as degradation ofthe CSPGs of a lesioned area of spinal cord or within the CNS, orrestoration, in whole or in part, of a motor, sensory or autonomicfunction of the mammal. Other embodiments of the invention are directedto inhibiting extravasation of cells. Yet other embodiments of theinvention are directed to enhancing or facilitating diffusion, asdiscussed herein. Other embodiments of the invention are directed totreating or preventing inflammation.

The terms “therapeutically effective amount” or “effective amount”, asused herein, may be used interchangeably and refer to an amount of atherapeutic compound component of the present invention. For example, atherapeutically effective amount of a therapeutic compound is apredetermined amount calculated to achieve the desired effect, i.e., toeffectively treat an injury to the central nervous system. For example,a therapeutic compound comprising a therapeutically effective amount ofa chondroitinase formulated to provide a stable, active enzyme, issufficient to degrade the CSPGs of a lesioned area of the spinal cord oran amount sufficient to restore, in whole or in part, a motor, sensoryor autonomic function of the mammal and may result in a regeneration ofneurons in a central nervous system, such as by promoting axonal growthinto an injured area. A therapeutically effective amount also includesan amount effective to degrade CSPGs and thereby promote recovery ofneurological function. A therapeutically effective amount also includesan amount sufficient to modify extravasation of cells or to reduce orprevent inflammation.

The terms “treat,” “treated,” or “treating” as used herein refers toboth therapeutic treatment and prophylactic or preventative measures,wherein the object is to prevent or slow down (lessen) an undesiredphysiological condition, disorder or disease, or to obtain beneficial ordesired clinical results. For the purposes of this invention, beneficialor desired clinical results include, but are not limited to, alleviationof symptoms; diminishment of the extent of the condition, disorder ordisease; stabilization (i.e., not worsening) of the state of thecondition, disorder or disease; delay in onset or slowing of theprogression of the condition, disorder or disease; amelioration of thecondition, disorder or disease state; and remission (whether partial ortotal), whether detectable or undetectable, or enhancement orimprovement of the condition, disorder or disease. Treatment includeseliciting a clinically significant response without excessive levels ofside effects. Treatment also includes prolonging survival as compared toexpected survival if not receiving treatment.

The process of “extravasation” is known as the transmigration of cells,such as leukocytes, from a blood vessel into the extravascular space,and may further include migration into surrounding tissue. As usedherein the term “leukocyte” is used to refer to the class of cellsassociated with inflammation, which may also be defined as any of thevarious blood cells that have a nucleus and cytoplasm. Also known aswhite blood cells, leukocytes include neutrophils, eosinophils,basophils, lymphocytes, such as B-cells, T-cells, monocytes andmacrophages. Four types of leukocytes are particularly important inimmune defense, including neutrophils, which release severalantibacterial proteins; monocytes, which are the precursors ofmacrophages that engulf and destroy foreign particles, and T and Blymphocytes, which are the antigen-recognizing cells of the immunecells.

The term “vector” refers to a vehicle which can transport the nucleicacid molecules. The nucleic acid molecules encoding the chondroitinasepolypeptide are covalently linked to the vector nucleic acid. With thisaspect of the invention, the vector can be, for example, a plasmid,single or double stranded phage, a single or double stranded RNA or DNAviral vector, or artificial chromosome, such as a BAC, PAC, YAC, OR MAC.

One embodiment of the present invention provides mutants ofchondroitinase ABCI. In a preferred embodiment, the chondroitinase ABCImutant enzymes and nucleic acids encoding them are those of the isolatedclones selected from 055D2-3 (deposited with American Type CultureCollection (ATCC), 10801 University Blvd., Manassas, Va. 20110-2209 onSep. 26, 2007 and having ATCC Deposit Designation PTA-8661) (SEQ ID NO:1and SEQ ID NO:9), 079B6-2 (deposited with ATCC on Sep. 26, 2007 andhaving ATCC Deposit Designation PTA-8662) (SEQ ID NO:2 and SEQ IDNO:10), 079D2-2 (deposited with ATCC on Sep. 26, 2007 and having ATCCDeposit Designation PTA-8659) (SEQ ID NO:3 and SEQ ID NO:11), 021B8-3(deposited with ATCC on Sep. 26, 2007 and having ATCC DepositDesignation PTA-8657), 057G1-1 (deposited with ATCC on Sep. 26, 2007 andhaving ATCC Deposit Designation PTA-8658) (SEQ ID NO:4 and SEQ ID NO:12), 023G6-4 (deposited with ATCC on Sep. 26, 2007 and having ATCCDeposit Designation PTA-8663) (SEQ ID NO:5 and SEQ ID NO:13) and005B12-3 (deposited with ATCC on Sep. 26, 2007 and having ATCC DepositDesignation PTA-8660) (SEQ ID NO: 6 and SEQ ID NO:14). The nucleotidesequence of chondroitinase ABCI is set forth as SEQ ID NO. 7 and theamino acid sequence of chondroitinase ABCI is set forth as SEQ ID NO. 8.

The ATCC deposits referred to herein will be maintained under the termsof the Budapest Treaty on the International Recognition of the Depositof Micro-Organisms for purposes of Patent Procedure. These deposits areprovided merely as convenience to those of skill in the art and are notan admission that a deposit is required under 35 U.S.C. §112. Thesequence(s) of the polynucleotides contained in the deposited materials,as well as the amino acid sequence of the polypeptides encoded thereby,are incorporated herein by reference and are controlling in the event ofany conflict with any description of sequences herein. A license may berequired to make, use or sell the deposited materials, and no suchlicense is hereby granted.

One embodiment of the present invention provides mutants ofchondroitinase ABCI. In preferred embodiments, such chondroitinase ABCImutant enzymes exhibit enhanced activity. In an embodiment, an enzyme ofthe invention has an enzyme activity level (as measured by its abilityto degrade a CSPG substrate) that is up to about two times greater thanthe activity level of the corresponding wild type enzyme. In anotherembodiment, an enzyme of the invention has an enzyme activity level thatis up to about three times greater than the activity of thecorresponding wild type chondroitinase. In an embodiment, thechondroitinase ABCI mutant enzymes are selected from 055D2-3 (SEQ IDNO:1), 079B6-2 (SEQ ID NO:2), 079D2-2 (SEQ ID NO:3), 057G1-1 (SEQ IDNO:4), 023G6-4 (SEQ ID NO:5) 005B12-3 (SEQ ID NO: 6), and 021B8-3 (ATCCDeposit Designation PTA-8657). More preferably, the enzyme is selectedfrom the group consisting of 055D2-3 (SEQ ID NO:1), 079B6-2 (SEQ IDNO:2), and 023G6-4 (SEQ ID NO:5).

The invention includes nucleic acids encoding the chondroitinase ABCImutant enzymes of the invention having enhanced activity, and methods oftheir use. In an embodiment, the invention includes nucleic acidsequences that encode the chondroitinase ABCI mutant enzymes selectedfrom 055D2-3 (SEQ ID NO:1), 079B6-2 (SEQ ID NO:2), 079D2-2 (SEQ IDNO:3), 057G1-1 (SEQ ID NO:4), 023G6-4 (SEQ ID NO:5) 005B12-3 (SEQ ID NO:6), and 021B8-3 (ATCC Deposit Designation PTA-8657). Preferably, anucleic acid sequence of the invention is selected from 055D2-3 nucleicacid (SEQ ID NO:9), 079B6-2 nucleic acid (SEQ ID NO:10), 079D2-2 nucleicacid (SEQ ID NO:11), 057G1-1 nucleic acid (SEQ ID NO:12), 023G6-4nucleic acid (SEQ ID NO:13) 005B12-3 nucleic acid (SEQ ID NO: 14), and021B8-3 (ATCC Deposit Designation PTA-8657).

In other preferred embodiments, such chondroitinase ABCI mutant enzymesexhibit enhanced resistance to inactivation. In an embodiment, enhancedresistance to inactivation permits an enzyme of the invention to remainactive following a stress (such as heat or UV) for a time that is up toabout ten-fold longer than for the corresponding wild typechondroitinase. For example, if a wild type chondroitinase maintainsmeasureable activity for up to about 3 days, a chondroitinase enzyme ofthe invention maintains measurable activity for up to about 30 daysunder the same conditions. In an embodiment, the chondroitinase ABCImutant enzymes having increased resistance to inactivation are selectedfrom 055D2-3 (SEQ ID NO:1), 079B6-2 (SEQ ID NO:2), 079D2-2 (SEQ IDNO:3), 057G1-1 (SEQ ID NO:4), 023G6-4 (SEQ ID NO:5) 005B12-3 (SEQ ID NO:6), and 021B8-3 (ATCC Deposit Designation PTA-8657). More preferably,the enzyme is selected from the group consisting of 055D2-3 (SEQ IDNO:1), 079B6-2 (SEQ ID NO:2), and 023G6-4 (SEQ ID NO:5).

The invention includes nucleic acids encoding the chondroitinase ABCImutant enzymes of the invention having enhanced resistance toinactivation, and methods of their use. In an embodiment, the inventionincludes nucleic acid sequences that encode the chondroitinase ABCImutant enzymes selected from 055D2-3 (SEQ ID NO:1), 079B6-2 (SEQ IDNO:2), 079D2-2 (SEQ ID NO:3), 057G1-1 (SEQ ID NO:4), 023G6-4 (SEQ IDNO:5) 005B12-3 (SEQ ID NO: 6), and 021B8-3 (ATCC Deposit DesignationPTA-8657). Preferably, a nucleic acid sequence of the invention isselected from 055D2-3 nucleic acid (SEQ ID NO:9), 079B6-2 nucleic acid(SEQ ID NO:10), 079D2-2 nucleic acid (SEQ ID NO:11), 057G1-1 nucleicacid (SEQ ID NO:12), 023G6-4 nucleic acid (SEQ ID NO:13) 005B12-3nucleic acid (SEQ ID NO: 14), and 021B8-3 (ATCC Deposit DesignationPTA-8657).

In a further embodiment, a mutant chondroitinase ABCI enzyme is providedhaving increased stability. The enzyme exhibits increased resistance toinactivation under stressed conditions, including exposure to UV lightor heat, as compared to that of wild-type ABCI enzyme. In a preferredembodiment, the enzyme exhibits increased stability compared towild-type chondroitinase ABCI enzyme following a challenge by a stress.In an embodiment, the chondroitinase ABCI mutant enzymes havingincreased stability are selected from 055D2-3 (SEQ ID NO:1), 079B6-2(SEQ ID NO:2), 079D2-2 (SEQ ID NO:3), 057G1-1 (SEQ ID NO:4), 023G6-4(SEQ ID NO:5) 005B12-3 (SEQ ID NO: 6), and 021B8-3 (ATCC DepositDesignation PTA-8657). More preferably, the enzyme is selected from thegroup consisting of 055D2-3 (SEQ ID NO:1), 079B6-2 (SEQ ID NO:2), and023G6-4 (SEQ ID NO:5).

The enzymes of the invention may be used to prevent, treat and alleviatesymptoms of inflammation and inflammatory states. In an embodiment, achondroitinase ABCI mutant enzyme of the invention is used to prevent,treat or alleviate symptoms of chronic inflammatory diseases. Achondroitinase ABCI mutant enzyme of the invention may be used to treatinflammation associated with pain, injection and diseased states. Anenzyme of the invention may be used to prevent tissue damage that isassociated with inflammatory processes. Several conditions, includingchronic inflammatory diseases, may benefit from controlled immuneresponse. Some examples of chronic inflammatory diseases include Asthma,Rheumatoid Arthritis (RA), Multiple Sclerosis (MS), Systemic LupusErythematosus (SLE), and Chronic Obstructive Pulmonary Disease (COPD).An enzyme of the invention may also be used to regulate the inflammatorystate associated with one or more disease selected from the groupconsisting of central nervous system disorders, central nervous systemdiseases, spinal cord injury, and cardiovascular diseases.

Inflammatory diseases, autoimmune diseases, and diseases with aninflammatory component that may be treated with a composition comprisingan enzyme of the invention also include Multiple Sclerosis, Meningitis,Encephalitis, Rheumatoid arthritis, Osteo arthritis, Lupus, Wegener'sgranulomatosis, Inflammatory bowel disease: Crohn's colitis, ulcerativecolitis, Asthma, Chlamydia infections, Syphilis, Thyroiditis, Temporalarteritis, Polymyalgia rheumatica, Ankylosing spondylitis, Psoriasis,Vasculitiditis such as: temporal arteritis, Takayasu arteritis,syphilitic aortitis, infectious aneurisms, atherosclerotic aneurisms,inflammatory abdominal aortic aneurysms, polyarteritis nodosa, Kawasakidisease, Churg-Strauss, hypersensitivity vasculitis, Buerger's disease,mesenteric inflammatory veno-occlusive disease, phlebitis,thrombophlebitis, Churg-Strauss, primary angiitis of the CNS, druginduced vasculitis, any secondary arteritis or venulitis, Gout,Pseudogout, Sarcoidosis, Sjogren's Syndrome, Myelitis, Salpingitis ofany etiology, Uveitis, Pelvic Inflammatory Disease, Glomerulonephritisof any etiology, Goodpasture's syndrome, Pericarditis, Myocarditis,Endocarditis, and Pancreatitis.

One embodiment of the present invention is a method for modifying accessof cells to extravascular spaces and regions comprising administering toa patient a composition comprising a chondroitinase ABCI mutant enzymeof the invention. Another embodiment of the present invention is amethod of reducing penetration of cells associated with inflammationinto tissue of a patient comprising administering to a patient acomposition comprising an enzyme of the invention.

Another embodiment of the invention is a method for inhibitingextravasation of cells associated with inflammation from blood vesselscomprising administering to a patient a composition comprising achondroitinase ABCI mutant enzyme of the invention. The enzyme of theinvention may prevent extravasation of cells selected from the groupselected from the group consisting of white blood cells, leukocytes,neutrophils, eosinophils, basophils, lymphocytes, B-cells, T-cells,monocytes, and macrophages cells from leaving the blood stream.

Another embodiment of the present invention is a method of treatinginflammation in a patient, the method comprising extracting circulatingcells from a patient, subjecting the cells to a chondroitinase ABCImutant enzyme of the invention ex vivo to modify the cells, andadministering the modified blood cells into the patient. Therefore, theuse of the enzymes described herein may also be directed to ex vivotreatments.

Extraction of cells may be accomplished by a variety of methodsincluding, but not limited to, intravenous blood withdrawal,transfusion, dialysis, bypass, organ transplant and other similarmethods that result in removal of cells from the body. Administration ofthe cells may be accomplished by the same methods used to extract thecells, including, but not limited to, intravenous administration,transfusion, dialysis, bypass, organ transplant and the like.

A circulating leukocyte with ligands expressed on its surface containingcarbohydrate chains may be extracted from a patient and modified ex vivoby one or more of the ABCI mutant enzymes of the invention. Extractionmay be accomplished by blood draw, transfusion, dialysis, bypass, ororgan transplant. As described, a chondroitinase ABCI mutant enzyme ofthe invention modifies the carbohydrate chains. Once modified, theleukocytes may be reintroduced into a patient's blood stream. Modifiedleukocytes will be incapable of adhering to endothelial expressedselectins, mucins, and integrins. Timing of an extraction andreintroduction into the bloodstream may be optimized by observing theinflammatory response and the appearance of leukocytes in the bloodstream, once said cells are signaled to specific sites of injury orinfection. As a result, extravasation of leukocytes into tissue may beregulated, prevented, reduced, or controlled. Such regulation may beused in methods and treatments as directed to control and treatinflammatory response and diseases with an inflammatory component.

The compositions of the present invention can be used for the treatmentof spinal cord injuries and in the promotion of regeneration of axons.The compositions of the present invention can also be used to promoteplasticity, regrowth, repair, and/or regeneration of dysfunctionalneurons in the CNS that have been damaged as a result of disease, suchas degenerative diseases including Alzheimer's and Parkinson's disease.Advantageously, the use of proteoglycan degrading polypeptides ormembrane transducing polypeptides in the compositions of the presentinvention also promote diffusion and access of damage or diseased tissueto other therapeutic agents promoting the regeneration of neurons.

A further embodiment of the present invention is a method of treatingcentral nervous system injuries comprising administering a compositioncomprising a chondroitinase ABCI mutant enzyme. In preferredembodiments, the chondroitinase ABCI mutant enzyme is administered in atherapeutically effective amount. In a preferred embodiment, thechondroitinase ABCI mutant enzyme used for treating central nervoussystem injuries is selected from the group consisting of 055D2-3 (SEQ IDNO:1), 079B6-2 (SEQ ID NO:2), 079D2-2 (SEQ ID NO:3), 057G1-1 (SEQ IDNO:4), 023G6-4 (SEQ ID NO:5) 005B12-3 (SEQ ID NO: 6), and 021B8-3 (ATCCDeposit Designation PTA-8657). More preferably, the enzyme is selectedfrom the group consisting of 055D2-3 (SEQ ID NO:1), 079B6-2 (SEQ IDNO:2), and 023G6-4 (SEQ ID NO:5). Such central nervous system injuriesmay include, but are not limited to, spinal cord injuries, includingtrauma induced injuries, contusions, or compress injuries.

Another embodiment of the present invention is a method promotingneuronal outgrowth comprising administering a composition comprising achondroitinase ABCI mutant enzyme. In preferred embodiments, thechondroitinase ABCI mutant enzyme is administered in a therapeuticallyeffective amount. In a preferred embodiment, the chondroitinase ABCImutant enzyme that promotes neuronal outgrowth is selected from thegroup consisting of 055D2-3 (SEQ ID NO:1), 079B6-2 (SEQ ID NO:2),079D2-2 (SEQ ID NO:3), 057G1-1 (SEQ ID NO:4), 023G6-4 (SEQ ID NO:5)005B12-3 (SEQ ID NO: 6), and 021B8-3 (ATCC Deposit DesignationPTA-8657). More preferably, the enzyme is selected from the groupconsisting of 055D2-3 (SEQ ID NO:1), 079B6-2 (SEQ ID NO:2), and 023G6-4(SEQ ID NO:5).

Other embodiments of the present invention relate to methods forpromoting neurological functional recovery after central nervous system(“CNS”) injury or disease. In preferred embodiments, the chondroitinaseABCI mutant enzyme is administered in a therapeutically effectiveamount. In particular, the present invention is directed to a method ofutilizing chondroitinase to promote sensory, motor or autonomicneurological functional recovery following injury in or to the spinalcord. Compositions useful in this method include acceptable formulationsof a chondroitinase ABCI mutant enzyme of the invention, including, forexample, immediate release and sustained release formulations of enzyme.The present invention is also directed to a method of promotingneurological functional recovery after a contusion injury to the spinalcord. The most common types of spinal cord injuries (SCI) includecontusions (bruising of the spinal cord) and compression injuries(caused by pressure on the spinal cord). In contusion injuries, the mostcommon type of injury, a cavity or hole often forms in the center of thespinal cord. The ABCI mutant enzymes of the invention can also be usedto degrade CSPGs. Accordingly, an embodiment of the invention includes amethod of degrading one or more CSPGs using a composition comprising anABCI mutant enzyme of the invention. Preferably a composition of theinvention effective for promoting neurological functional recoverycomprises a chondroitinase ABCI mutant enzyme selected from 055D2-3 (SEQID NO:1), 079B6-2 (SEQ ID NO:2), 079D2-2 (SEQ ID NO:3), 057G1-1 (SEQ IDNO:4), 023G6-4 (SEQ ID NO:5) 005B12-3 (SEQ ID NO: 6), and 021B8-3 (ATCCDeposit Designation PTA-8657).

One embodiment of the present invention is a composition and a methodfor its use that facilitates the access and distribution of atherapeutic and diagnostic agent in the composition into cells, throughmembranes or into tissues by the use of composition that includes atleast one enzyme capable of cleaving proteoglycans. Preferably thecomposition comprises a chondroitinase ABCI mutant enzyme selected from055D2-3 (SEQ ID NO:1), 079B6-2 (SEQ ID NO:2), 079D2-2 (SEQ ID NO:3),057G1-1 (SEQ ID NO:4), 023G6-4 (SEQ ID NO:5) 005B12-3 (SEQ ID NO: 6),and 021B8-3 (ATCC Deposit Designation PTA-8657). The molecules or agentsin the composition may include one or more of growth factors including,for example, Brain Derived Neurotrophic Factor, Insulin-like GrowthFactor, Fibroblast Growth Factor, Ciliary Neurotrophic Factor, GlialDerived Neurotrophic Factor, Transforming Growth Factor, Glial GrowthFactor 2, L1, GM1, Vascular Endothelial Growth Factor, Nerve GrowthFactor, and Immunophilins. The composition in some embodiments comprisesa fluorescent or contrast agent for imaging. According to an embodiment,the agent includes a cell for transplant, for example a stem cell orneuron, a cell as a delivery agent, a chemotherapeutic agent, anantibiotic, an antibody, or a Nogo receptor antagonist. The compositionscan be used for treating a CNS injury. Preferably the composition isused in the treatment of neuronal damage from a contusion injury.

The treatments described herein deliver an amount of a chondroitinaseABCI mutant enzyme effective to degrade CSPGs and thereby promote, forexample, the recovery of neurological function, optionally including atherapeutic agent, to the CNS. Such methods may include optionallyadministering, in combination with a chondroitinase ABCI mutant enzymeof the invention, another chondroitinase, including, but not limited tochondroitinase ABCI, chondroitinase ABCII, chondroitinase AC andchondroitinase B or co-administering a mammalian enzyme withchondroitinase-like activity, such as hyaluronidases Hyal 1, Hyal2,Hyal3, Hyal4, and PH20 preferably to the CNS, and more preferably to thelesions of the injured area of the CNS. Once the proteins orpolypeptides in the compositions have been purified to the extentdesired, they may be suspended or diluted in an appropriatephysiological carrier or excipient for treatment.

Chondroitinase may be obtained from various sources, including amicroorganism that naturally expresses a chondroitinase; for example,but not limited to, E. coli, Proteus vulgaris, or from the expression ofa recombinant protein in a host cell. The host cell can be a prokaryoticcell (such as E. coli) or a eukaryotic cell (such as yeast, a mammaliancell or an insect cell).

The chondroitinase ABCI mutant nucleic acids of the present inventionmay be obtained by a number of methods known in the art. For example,one may use the polymerase chain reaction and/or other techniques togenerate mutations in the wild type P. vulgaris or other chondroitinaseencoding sequence. In an embodiment, the invention includes a method ofmaking a nucleic acid sequence that encodes a chondroitinase ABCI mutantenzyme selected from 055D2-3 (SEQ ID NO:1), 079B6-2 (SEQ ID NO:2),079D2-2 (SEQ ID NO:3), 057G1-1 (SEQ ID NO:4), 023G6-4 (SEQ ID NO:5)005B12-3 (SEQ ID NO: 6), and 021B8-3 (ATCC Deposit DesignationPTA-8657). Preferably, the invention includes a method of making anucleic acid sequence of the invention, wherein the nucleic acid isselected from 055D2-3 nucleic acid (SEQ ID NO:9), 079B6-2 nucleic acid(SEQ ID NO:10), 079D2-2 nucleic acid (SEQ ID NO:11), 057G1-1 nucleicacid (SEQ ID NO:12), 023G6-4 nucleic acid (SEQ ID NO:13) 005B12-3nucleic acid (SEQ ID NO: 14), and 021B8-3 (ATCC Deposit DesignationPTA-8657).

Expression of a recombinant ABCI mutant nucleic acid sequence of theinvention can be performed by ligating a nucleic acid encoding the ABCImutant protein, or a portion thereof, into a vector suitable forexpression in either prokaryotic cells, eukaryotic cells, or both.Procedures for ligation are well known to those of ordinary skill in theart. Expression vectors for production of recombinant forms of thesubject chondroitinase polypeptides include plasmids and other vectors.For instance, suitable vectors for the expression of a chondroitinaseABCI mutant polypeptide include plasmids of the types: pBR322-derivedplasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derivedplasmids and pUC-derived plasmids for expression in prokaryotic cells,such as E. coli.

A number of vectors exist for the expression of recombinant proteins inyeast and could be used to express a recombinant ABCI mutant protein ofthe invention. For instance, YEP24, YIP5, YEP51, YEP52, pYES2, and YRP17are cloning and expression vehicles useful in the introduction ofgenetic constructs into S. cerevisiae (see, for example, Broach et al.(1983) in Experimental Manipulation of Gene Expression, ed. M. InouyeAcademic Press, p. 83, incorporated by reference herein).

In another embodiment, a chondroitinase ABCI mutant polypeptide of theinvention is produced recombinantly utilizing an expression vectorgenerated by subcloning the coding sequence of one of the chondroitinaseproteins represented in 055D2-3 (SEQ ID NO:1), 079B6-2 (SEQ ID NO:2),079D2-2 (SEQ ID NO:3), 057G1-1 (SEQ ID NO:4), 023G6-4 (SEQ ID NO:5)005B12-3 (SEQ ID NO: 6), and 021B8-3 (ATCC Deposit DesignationPTA-8657).

In some instances, it may be desirable to express a recombinantchondroitinase ABCI mutant polypeptide of the invention by the use of aninsect expression system such as the baculovirus expression system.Examples of such baculovirus expression systems include pVL-derivedvectors (such as pVL1392, pVL1393 and pVL941), pAcUW-derived vectors(such as pAcUW1), and pBlueBac-derived vectors (such as the β-galcontaining pBlueBac III).

The expression vectors and host cells listed herein are provided by wayof example only and represent the well-known systems available to thoseof ordinary skill in the art that may be useful to express the nucleicacid molecules. The person of ordinary skill in the art would be awareof other systems suitable for maintenance propagation or expression ofthe nucleic acid molecules described herein.

The enzymes of the invention may be formulated into pharmaceuticalcompositions and formulations. Suitable stable formulations and methodsof purification are set forth in co-pending PCT Application No.US2005/017464 filed May 18, 2005 entitled “Methods of PurifyingChondroitinase and Stable Formulations Thereof” herein incorporated byreference in its entirety.

Various embodiments provide a stable formulation of a chondroitinaseABCI mutant enzyme of the invention for both storage and administration.Generally, the enzyme of such stable formulations exhibit at least about50% of activity at about 24 hours, preferably at least about 75% ofactivity, more preferably at least about 85% of activity. In anotheraspect of the invention, the formulations consistently provide stablechondroitinase activity.

In one embodiment, the chondroitinase is formulated in a phosphatebuffer, preferably a sodium phosphate buffer with a concentration in therange of about 50 mM to about 1 M. A preferred embodiment is about 750mM sodium phosphate. Another preferred embodiment is about 100 mM sodiumphosphate. In a further embodiment the chondroitinase may be formulatedin a sodium phosphate buffer that further comprises sodium acetate.Sodium acetate may be present in the range of 25 mM to about 75 mM. In apreferred embodiment the sodium acetate concentration is about 50 mM. Inone embodiment a preferred formulation for administration is achondroitinase in a buffer with a pH of about 7.4. Further embodimentsof formulations for storage and administration are provided in theExamples described.

In further embodiments, a formulation comprising a purifiedchondroitinase ABCI mutant enzyme of the invention and a buffercomprising an increased ionic strength is provided. Embodiments whereina formulation comprises an increased ionic concentration may increasestability of an enzyme formulation. For example, a preferred embodimentprovides a formulation with about 1 M NaCl in sodium phosphate. Theconcentration of sodium phosphate may be about 50 mM. In a preferredembodiment, the enzyme storage concentration is below about 0.4 mg/ml.

In one embodiment, a chondroitinase ABCI mutant enzyme formulationcomprises about 0.4 mg/ml of a chondroitinase ABCI mutant enzyme of theinvention in about 100 mM Na phosphate, at a pH of about 7.4 with apreferred substrate specificity for chondroitin A, B, and C about thesame.

Various embodiments provide a stable formulation of a chondroitinaseABCI mutant enzyme of the invention for both storage and administration.Generally, the enzyme of such stable formulations exhibit at least about50% of activity at about 24 hours, preferably at least about 75% ofactivity, more preferably at least about 85% of activity. In anotheraspect of the invention, the formulations consistently provide stablechondroitinase activity.

In another embodiment, a chondroitinase ABCI mutant enzyme purificationis provided comprising the following steps: 1) extracting the enzymefrom a cell, 2) separating the crude cell extract using cation-exchangechromatography, 3) further separating the extract by a gel filtrationchromatography, and 4) removing endotoxin through an anion-exchangemembrane to produce a purified chondroitinase ABCI mutant enzyme of theinvention. In an embodiment a purified chondroitinase ABCI of theinvention is dialyzed into a volatile buffer, lyophilized and stored at−80° C.

Chondroitinase activity can be stabilized by the addition of excipientsor by lyophilization. Stabilizers include carbohydrates, amino acids,fatty acids, and surfactants and are known to those skilled in the art.Examples include carbohydrates such as sucrose, lactose, mannitol, anddextran, proteins such as albumin and protamine, amino acids such asarginine, glycine, and threonine, surfactants such as TWEEN® andPLURONIC®, salts such as calcium chloride and sodium phosphate, andlipids such as fatty acids, phospholipids, and bile salts.

Chondroitinase ABCI mutant enzymes of the invention may be administeredtopically, locally or systemically. Topical or local administration ispreferable for greater control of application. An enzyme of theinvention, singularly or in combination with other enzymes of theinvention or with other CSPG-degrading enzymes, can be mixed with anappropriate pharmaceutical carrier prior to administration.Administration includes delivery of the enzyme to the site of injury orsite at which CSPGs to be degraded are found. Examples of generally usedpharmaceutical carriers and additives are conventional diluents,binders, lubricants, coloring agents, disintegrating agents, bufferagents, isotonizing fatty acids, isotonizing agents, preservants,anesthetics, surfactants and the like, and are known to those skilled inthe art. Pharmaceutical carriers that may be used include dextran,sucrose, lactose, maltose, xylose, trehalose, mannitol, xylitol,sorbitol, inositol, serum albumin, gelatin, creatinine, polyethleneglycol, non-ionic surfactants (e.g. polyoxyethylene sorbitan fatty acidesters, polyoxyethylene hardened castor oil, sucrose fatty acid esters,polyoxyethylene polyoxypropylene glycol) and similar compounds.

A treatment regimen according to the invention may be carried out by ameans of administering a composition comprising a chondroitinase ABCImutant enzyme of the present invention. The treatment regimen mayfurther comprise administering chondroitinase ABCI, chondroitinaseABCII, chondroitinase AC and chondroitinase B or mammalian enzymes withchondroitinase-like activity such as hyaluronidases Hyall, Hyal2, Hyal3,Hyal4 and PH20 to the lesions of the injured area of the CNS. The modeof administration, the timing of administration and the dosage arecarried out such that the functional recovery from impairment of the CNSis enhanced by the promotion of neurite outgrowth.

The effective amount of chondroitinase can be administered in a singledosage, two dosages or a plurality of dosages. Although it is to beunderstood that the dosage may be administered at any time, in oneembodiment, the dosage is administered within 12 hours after injury, oras soon as is feasible. In another embodiment, the dosage isadministered to an injured mammal in one, two or a plurality of dosages;such dosages would be dependant on the severity of the injury and theamount of CSPGs present in the glial scarring. Where a plurality ofdosages is administered, they may be delivered on a daily, weekly, orbi-weekly basis. The delivery of the dosages may be by means of catheteror syringe. Alternatively, the treatment can be administered duringsurgery to allow direct application to the glial scar.

For example, in some aspects, the invention is directed to apharmaceutical composition comprising a compound, as defined above, anda pharmaceutically acceptable carrier or diluent, or an effective amountof a pharmaceutical composition comprising a compound as defined above.

The compounds of the present invention can be administered in theconventional manner by any route where they are active. Administrationcan be systemic, topical, or oral. For example, administration can be,but is not limited to, parenteral, subcutaneous, intravenous,intramuscular, intraperitoneal, transdermal, oral, buccal, or ocularroutes, or intravaginally, by inhalation, by depot injections, or byimplants. Thus, modes of administration for the compounds of the presentinvention (either alone or in combination with other pharmaceuticals)can be, but are not limited to, sublingual, injectable (includingshort-acting, depot, implant and pellet forms injected subcutaneously orintramuscularly), or by use of vaginal creams, suppositories, pessaries,vaginal rings, rectal suppositories, intrauterine devices, andtransdermal forms such as patches and creams.

Specific modes of administration will depend on the indication. Theselection of the specific route of administration and the dose regimenis to be adjusted or titrated by the clinician according to methodsknown to the clinician in order to obtain the optimal clinical response.The amount of compound to be administered is that amount which istherapeutically effective. The dosage to be administered will depend onthe characteristics of the subject being treated, e.g., the particularanimal treated, age, weight, health, types of concurrent treatment, ifany, and frequency of treatments, and can be easily determined by one ofskill in the art (e.g., by the clinician).

Pharmaceutical formulations containing the compounds of the presentinvention and a suitable carrier can be solid dosage forms whichinclude, but are not limited to, tablets, capsules, cachets, pellets,pills, powders and granules; topical dosage forms which include, but arenot limited to, solutions, powders, fluid emulsions, fluid suspensions,semi-solids, ointments, pastes, creams, gels and jellies, and foams; andparenteral dosage forms which include, but are not limited to,solutions, suspensions, emulsions, and dry powder; comprising aneffective amount of a polymer or copolymer of the present invention. Itis also known in the art that the active ingredients can be contained insuch formulations with pharmaceutically acceptable diluents, fillers,disintegrants, binders, lubricants, surfactants, hydrophobic vehicles,water soluble vehicles, emulsifiers, buffers, humectants, moisturizers,solubilizers, preservatives and the like. The means and methods foradministration are known in the art and an artisan can refer to variouspharmacologic references for guidance. For example, ModernPharmaceutics, Banker & Rhodes, 4^(th) Ed., Informa Healthcare (2002);and Goodman & Gilman's The Pharmaceutical Basis of Therapeutics, 10thEd., McGraw-Hill (2001) can be consulted.

The compounds of the present invention can be formulated for parenteraladministration by injection, e.g., by bolus injection or continuousinfusion. The compounds can be administered by continuous infusionsubcutaneously over a period of about 15 minutes to about 24 hours.Formulations for injection can be presented in unit dosage form, e.g.,in ampoules or in multi-dose containers, with an added preservative. Thecompositions can take such forms as suspensions, solutions or emulsionsin oily or aqueous vehicles, and can contain formulatory agents such assuspending, stabilizing and/or dispersing agents.

For oral administration, the compounds can be formulated readily bycombining these compounds with pharmaceutically acceptable carriers wellknown in the art. Such carriers enable the compounds of the invention tobe formulated as tablets, pills, dragees, capsules, liquids, gels,syrups, slurries, suspensions and the like, for oral ingestion by apatient to be treated. Pharmaceutical preparations for oral use can beobtained by adding a solid excipient, optionally grinding the resultingmixture, and processing the mixture of granules, after adding suitableauxiliaries, if desired, to obtain tablets or dragee cores. Suitableexcipients include, but are not limited to, fillers such as sugars,including, but not limited to, lactose, sucrose, mannitol, and sorbitol;cellulose preparations such as, but not limited to, maize starch, wheatstarch, rice starch, potato starch, gelatin, gum tragacanth, methylcellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose,and polyvinylpyrrolidone (PVP). If desired, disintegrating agents can beadded, such as, but not limited to, the cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodiumalginate.

Dragee cores can be provided with suitable coatings. For this purpose,concentrated sugar solutions can be used, which can optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments can be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical preparations which can be used orally include, but arenot limited to, push-fit capsules made of gelatin, as well as soft,sealed capsules made of gelatin and a plasticizer, such as glycerol orsorbitol. The push-fit capsules can contain the active ingredients inadmixture with filler such as, e.g., lactose, binders such as, e.g.,starches, and/or lubricants such as, e.g., talc or magnesium stearateand, optionally, stabilizers. In soft capsules, the active compounds canbe dissolved or suspended in suitable liquids, such as fatty oils,liquid paraffin, or liquid polyethylene glycols. In addition,stabilizers can be added. All formulations for oral administrationshould be in dosages suitable for such administration.

For buccal administration, the compositions can take the form of, e.g.,tablets or lozenges formulated in a conventional manner.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebulizer, with the useof a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitcan be determined by providing a valve to deliver a metered amount.Capsules and cartridges of, e.g., gelatin for use in an inhaler orinsufflator can be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

The compounds of the present invention can also be formulated in rectalcompositions such as suppositories or retention enemas, e.g., containingconventional suppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds ofthe present invention can also be formulated as a depot preparation.Such long acting formulations can be administered by implantation (forexample subcutaneously or intramuscularly) or by intramuscularinjection.

Depot injections can be administered at about 1 to about 6 months orlonger intervals. Thus, for example, the compounds can be formulatedwith suitable polymeric or hydrophobic materials (for example as anemulsion in an acceptable oil) or ion exchange resins, or as sparinglysoluble derivatives, for example, as a sparingly soluble salt.

In transdermal administration, the compounds of the present invention,for example, can be applied to a plaster, or can be applied bytransdermal, therapeutic systems that are consequently supplied to theorganism.

Pharmaceutical compositions of the compounds also can comprise suitablesolid or gel phase carriers or excipients. Examples of such carriers orexcipients include but are not limited to calcium carbonate, calciumphosphate, various sugars, starches, cellulose derivatives, gelatin, andpolymers such as, e.g., polyethylene glycols.

The compounds of the present invention can also be administered incombination with other active ingredients, such as, for example,adjuvants, protease inhibitors, or other compatible drugs or compoundswhere such combination is seen to be desirable or advantageous inachieving the desired effects of the methods described herein.

The following methods are used to illustrate the various embodiments ofthe present invention. The methods are exemplary methods and are notmeant to limit the invention.

EXAMPLE 1

The present example illustrates the generation of exemplarychondroitinase ABCI mutant enzymes and nucleic acids according to thepresent invention.

Cloning of wildtype cABCI: Chondroitinase ABCI was generated by PCRusing the full-length cDNA from P. vulgaris and cloned in the pET15bexpression vector at the NdeI and BamHI sites. The vector was expressedin E. coli (Prabhakar V, et al. Biochem J. 2005).

Random mutagenesis of cABCI: The chondroitinase ABCI gene was dividedinto four modules. Random mutagenesis was performed on each individualmodule, using the Genemorph II kit (Stratagene) to create a productcontaining 1-2 amino acid changes per mutant. Products were cloned andtransformed into E. coli DH10B such that the number of colonies obtainedcontaining the correct clone structure was at least 5-times the numberof individual mutant genes predicted to exist in the DNA population. Thecolonies were pooled and plasmid DNA was purified and used to transformthe expression strain, BL21.

Thermal stress assay: E. coli strains expressing the mutated cABCIenzymes were clonally plated for growth and induction (OvernightExpress, Novagen) in 96-well plates. E. coli expressing the wildtypeenzyme were also included. Total protein was extracted from theresulting bacterial pellets using BPER (PIERCE) followed by a 1:50dilution with PBS. Samples were subjected to a thermal stress of 42degrees C. in a humidified incubator for 2 hours. Samples were thenmixed with an equal volume of 0.25 mg/ml chondroitin sulfate C (Sigma),a substrate of cABCI which results in the cleavage of the GAG chains.After a 10 minute room temperature incubation, DMB reagent was added,and absorbance at 660 nm was measured. Positive hits with absorbancemeasurements greater than the wildtype enzyme on the same plate werecounted as positive hits, indicating greater activity after thermalstress.

Creation of recombined library: The ten most thermal resistant clonesfrom modules A, B and C were recombined in a random fashion to produce acombinatorial product library. The PCR products from each module werecombined in an equimolar ratio, with one molar equivalent of thecorresponding wild type also present. This created a pool of 9 variantsequences for Module C, and a pool of 11 variants for both Modules A andB. A 3-way ligation was performed in which each module could only beligated in the correct orientation with the appropriate flankingmodule(s) and ligated into pET15b vector DNA to produce expressionclones containing full-length cABCI. The total size of this library is1089 variant cABCI sequences. The number of colonies obtained containingthe correct clone structure was at least 5-times the number ofindividual mutant genes predicted. The ligation was weighted to mostlyproduce clones containing two or three mutant modules, thereby creatingnew combinations of the mutations identified in the initial screening“hits.”

EXAMPLE 2

The present example illustrates exemplary chondroitinase mutant enzymesof the present invention.

Thermally stable mutants were confirmed to be generated through theprocess of molecular evolution. The modified DMB assay identified cloneswith greater thermal stability at 42 degrees C. for 2 hours whencompared to wildtype cABCI. Stability at this temperature is likely toconfer greater stability at 37 degrees C., enabling ease of handling anddelivery for in vivo studies, as indwelling mini-pumps could be utilizedfor dosing. Individual modules resulted in an expected range of positivehits overall as defined by study parameters.

Clones having increased thermal stability were characterized bysequencing. All nucleotide and amino acid sequences are indicated as thewild-type and then the mutant version (Wild-type to Mutant).

Chondroitinase Chondroitinase ABCI mutant Amino Acid ABCI mutantNucleotide enzyme (protein) sequence (nucleic acid) sequence 055D2-3protein E256 to K256 055D2-3 nucleic T450 to C450 (SEQ ID NO. 1) acidG766 to A766 (SEQ ID NO. 9) C2295 to T2295 079B6-2 protein D683 to N683079B6-2 nucleic G2047 to A2047 (SEQ ID NO. 2) acid (SEQ ID NO. 10)079D2-2 protein 079D2-2 nucleic A1773 to G1773 (SEQ ID NO. 3) acid G1980to A1980 (SEQ ID NO. 11) T2068 to C2068 A2076 to G2076 057G1-1 protein057G1-1 nucleic G483 to A483 (SEQ ID NO. 4) acid T1110 to C1110 (SEQ IDNO. 12) T1821 to C1821 023G6-4 protein I919 to F919 023G6-4 nucleicA2755 to T2755 (SEQ ID NO. 5) A736 to P736 acid G2206 to C2206 (SEQ IDNO. 13) 005B12-3 protein E296 to K296 005B12-3 nucleic G985 to A985 (SEQID NO. 6) acid (SEQ ID NO. 14)

EXAMPLE 3

Stability Assessment--Bacterial Lysate 37° C. Stability. Wildtype andvariant chondroitinase ABCI expressing E. coli were expanded andexpressed in 96-well plates. Protein extracts were prepared from theresulting bacterial pellets. Pellets were lysed with BPER (Pierce) forten minutes at room temperature and spun at 1000g to pellet anyunsolublized material. The supernatants were transferred to newcontainers. Protein content was normalized using a BCA protein assay.Lysates were also run on SDS-PAGE gels and Coomassie stained. The amountof enzyme produced was measured using GeneTools software (Syngene)comparing the size of the enzyme band to all other extracted proteinbands (histogram copied in at end of document.) Percent of enzyme on thebasis of total cell lysate protein is shown in FIG. 1.

Samples were subjected to a thermal stress of 37 degrees C. in ahumidified incubator. Activity was measured incrementally over timeusing a colorimetric DMB (Dimethyl, methylene blue) assay. Samples weremixed with an equal volume of 0.25 mg/ml chondroitin sulfate C, asubstrate of chondroitinase ABCI which results in the cleavage of theGAG chains. After a ten minute room temperature incubation DMB reagentwas added and the absorbance at 660 nm was measured. Results aredepicted in FIG. 2 and in Tables 1A and 1B below.

TABLE 1A Time (days) at 37 degrees C. 0.00 0.71 1.00 1.65 2.81 3.69 4.699.69 11.69 057G1-1 0.94 1.04 0.88 1.05 0.90 1.18 1.04 0.90 0.69 023G6-40.94 1.06 1.03 1.05 0.92 1.22 1.04 1.04 1.08 005B12-3 0.97 1.08 0.911.10 0.93 1.24 1.11 1.09 0.97 079D2-2 0.93 1.06 0.92 1.05 0.90 1.18 1.020.85 0.72 079B6-2 0.93 1.08 0.89 1.05 1.05 1.18 1.07 1.12 1.12 021B8-30.97 1.07 0.91 1.08 0.95 1.22 1.08 1.07 0.92 055D2-3 0.92 1.03 0.86 1.060.92 1.15 1.04 1.03 0.92 +cABC1 0.95 1.07 1.02 1.09 0.18 0.09 0.03 0.08−0.03

TABLE 1B Time (days) at 37 degrees C. (continued) 13.69 16.69 18.6920.69 23.69 25.69 27.69 31.69 057G1-1 0.30 0.20 0.19 −0.01 0.06 0.090.11 0.08 023G6-4 1.00 0.95 0.60 0.01 0.09 0.16 0.18 0.05 005B12-3 0.630.49 0.46 0.02 0.02 0.03 0.09 0.15 079D2-2 0.32 0.28 0.32 0.06 0.18 0.230.09 0.08 079B6-2 1.07 1.07 0.94 0.73 0.38 0.42 0.27 0.14 021B8-3 0.420.19 0.15 0.08 0.22 0.10 0.10 0.03 055D2-3 0.57 0.21 0.29 0.30 0.21 0.140.14 0.11 +cABC1 0.02 −0.13 0.06 −0.01 0.05 0.06 0.09 0.05

EXAMPLE 4

Semi-Purified 37° C. Stability. All enzymes from wildtype and variantchondroitinase ABCI expressing E. coli were purified using a high speedSP column. Protein samples were normalized by A280 to match theabsorbance reading of the native enzyme (0.35) by dilution in elutionbuffer (20 mM NaAcetate+250 mM NaCl). A fully purified cABCI enzyme wasalso reconstituted and diluted to an A280 of 0.35 same as the nativesemi-purified sample. Initial activity readings were taken for allsamples using a chondroitin C substrate spectrophotometric assay. Theassay measures the product produced by the digestion of chondroitinsulfate C over time at A232. Samples were subjected to a thermal stressof 37 degrees C. in a humidified incubator. Activity readings were takenevery day until the native sample lost all activity. Assaying ofremaining samples continued 3 times a week. Activity readings displayedas percent of total activity retained for a few variants are presentedin FIG. 3 and in Tables 2A and 2B below.

TABLE 2A Time (days) at 37 d C. 0.50 1.50 2.50 3.79 4.79 5.79 cABCI56.43 34.45 25.48 14.42 8.63 5.77 023G6-1 58.32 42.28 41.70 31.96 24.6119.82 079B6-2 65.23 54.21 44.96 35.14 19.09 17.93 cABCI- 33.78 18.6411.21 4.51 1.59 0.66 Purified

TABLE 2B 6.79 8.79 11.79 13.79 15.79 18.79 cABCI 3.55 2.23 0.74 0.000.00 0.00 023G6-1 15.23 12.41 9.00 6.74 3.76 1.86 079B6-2 13.89 10.765.02 2.89 1.51 1.10 cABCI- 0.00 0.00 0.00 0.00 0.00 0.00 Purified

EXAMPLE 5

Stability Of Mutant Chondroitinase Following UV Treatment. After growthand expression, a chondroitinase mutant is extracted using BPER (Pierce)as above and exposed to UV light. The chondroitin lyase activity ismeasured by a DMB assay.

The present invention is not limited to the embodiments described andexemplified above, but is capable of variation and modification withinthe scope of the appended claims.

What is claimed is:
 1. A pharmaceutical composition comprising aneffective amount of mutant chondroitinase ABC I enzyme selected from SEQID NO: 1, SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO:6, and combinationsthereof, and a pharmaceutically acceptable carrier or diluent.
 2. Thepharmaceutical composition of claim 1, wherein the composition comprisesmutant chondroitinase ABC I enzyme SEQ ID NO:
 1. 3. The pharmaceuticalcomposition of claim 1, wherein the composition comprises mutantchondroitinase ABC I enzyme SEQ ID NO:
 2. 4. The pharmaceuticalcomposition of claim 1, wherein the composition comprises mutantchondroitinase ABC I enzyme SEQ ID NO:
 5. 5. The pharmaceuticalcomposition of claim 1, wherein the composition comprises mutantchondroitinase ABC I enzyme SEQ ID NO:
 6. 6. The pharmaceuticalcomposition of claim 1, wherein the composition is a sustained releasecomposition.
 7. A pharmaceutical composition comprising an effectiveamount of a nucleic acid sequence encoding for a mutant chondroitinaseABC I enzyme, wherein the nucleic acid sequence is selected from SEQ IDNO: 9, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO:14, and combinationsthereof, and a pharmaceutically acceptable carrier or diluent.
 8. Thepharmaceutical composition of claim 7, wherein the composition comprisesthe nucleic acid sequence of SEQ ID NO:
 9. 9. The pharmaceuticalcomposition of claim 7, wherein the composition comprises the nucleicacid sequence of SEQ ID NO:
 10. 10. The pharmaceutical composition ofclaim 7, wherein the composition comprises the nucleic acid sequence ofSEQ ID NO:
 13. 11. The pharmaceutical composition of claim 7, whereinthe composition comprises the nucleic acid sequence of SEQ ID NO: 14.12. The pharmaceutical composition of claim 7, wherein the compositionis a sustained release composition.